Alternative .splicing allows hutTians to protiuce a vast proteome diversity from a relatively few (~24,000) protein coding genes. This ROO proposal is part: of an overall objective to gain insight into the fole of alternative splicing in retinal development. 1 have chosen to address this question by focusing on SfrslO, with the specific hypothesis that it is essential for cell fate determination and differentiation. The overall goal is to determine the expression, function and targets of SfrslO during retinal development. I have employed in situ hybridization, immunofluorescence and microarray analysis to determine the expression of SfrslO during retinal development. In all, SfrslO appears to be expressed in progenitor cells and differentiating amacrine cells. Interestingly, the microarray data obtained from single retinal cells shows that certain progenitor cell markers such as cycling Dl and Sfrsp2 are enriched along with the expression of SfrslO. To determine the function of SfrslO,! have employed in vivo electroporation to deliver F^NAi or the misexpression construct in PO retinal progenitor cells. 1 have found that loss of SfrslO function results in increase in the numberof Mullerglia at the expense of neurons. This experiment utilizes a Hamilton needle to deliver the plasmid into the subretinal space. However, this method has often created damage to the retina. 1 have now employed a different method which utilized glass needles that leave a negligible imprint on the retina. While RNAi constructs reported in the grant have been used to determine the function of SfrslO, a conditional knockout mouse is crucial to this proposal. The production of such a mouse has been initiated at the University of Connecticut, where I will begin my position as an assistant professor on August I'S 2010. As 1 continue this project in my laboratory at the University of Connecticut, I will further refine the expression of SfrslO, perform gain and loss of SfrslO function with the electroporation techinque. Finally, the conditional knockout mouse will be crossed to retina specific Cre lines followed by detailed analysis of the phenotype. Also, the knockout mouse retinal cells will be used for deep (454) sequecing to identify the targets of SfrslO. Extenstion of this work in my laboratory will provide me with preliminary data that 1 intend to use for an ROl grant that should launch my independent career in science.